Fragments of prion proteins

ABSTRACT

Synthetic polypeptides having at least one antigenic site of a prion protein, methods for their use and manufacture, antibodies raised against such polypeptides and diagnostic kits containing these polypeptides or antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is a division of application Ser. No. 08/244,701 filedJun. 2, 1994, now U.S. Pat. No. 5,773,572, issued Jun. 30, 1998 whichwas a national stage filing under 35 USC 371 of PCT/GB92/02246, filedDec. 3, 1992.

The present invention relates to synthetic polypeptides. In particularit relates to synthetic polypeptides which emulate the three-dimensionalstructures and/or electrostatic surfaces and/or other physical, chemicaland structural properties of specific regions of proteins thought to bethe involved in the molecular pathology of spongiform encephalopathies.It is of particular interest to the design of immunodiagnostics,vaccines and other medical, veterinary or scientific agents in relationto human, bovine and ovine spongiform encephalopathies.

Spongiform encephalopathies are a group of, degenerative neurologicaldiseases. Examples have been found in a number of species includingsheep (where it is known as scrapie), cows (BSE) and humans(Creutzfeldt-Jakob disease (CJD) and kuru) (Review article, Taylor, D.M. Veterinary Record 125,413-415 (1989)). Similar conditions have alsobeen found in the wild mink population and in captive kudus (a kind ofantelope) and tigers. It has been variously reported that BSE can betransmitted under laboratory conditions to mice and pigs. This crossingof species barriers by the infective agent has led to increased concernthat transfer to humans could occur.

These diseases are characterised by a slow incubation time of four tofive years after which the clinical symptoms of progressive degenerationof mental state, including aggressiveness and lack of coordination,appear. Post mortems reveal a characteristic pattern of vacuolation inbrain tissue due to the destruction of neural cells, and the depositionof unusual protein fibres.

Although the form of the disease found in sheep (scrapie) has been knownfor many years, spongiform encephalopathies have come to prominencewithin the last decade following the appearance of BSE in cattle farms.The incidence of BSE in the United Kingdom has increased markedly duringthis period and public concern over the possible transmission of thedisease to humans has led to a collapse in the beef market. Thus forboth veterinary and economic reasons, there is an urgent need fordiagnostic agents to detect infection and for vaccines to preventinfection.

It is believed that the causative agent of scrapie and its counterpartsin other animals is a so-called “prion”, that is an infective particlecomprising protein only and no nucleic acid, the presence of the latterbeing required in the case of a conventional virus. In scrapie, oneparticular protein (termed prion protein, PrP^(sc)) has been found toco-purify with infectivity and can produce a scrapie-like condition inbrain cell cultures from other animals, such as hamsters, underlaboratory conditions. PrP^(sc) is the only known component of thecharacteristic protein fibres deposited in the brain tissue ofscrapie-infected sheep. The term “PrP^(sc)” as used herein should betaken to refer not only to the specific Prion protein identified insheep but also to those homologous proteins found in many other specieswhich appear to undergo a structural modification as describedhereinafter. The term “PrP^(c)” shall be used in respect of the normalcellular counterpart to PrP^(sc).

The major problem in the search for a specific diagnostic agent orsynthetic vaccine against the scrapie agent PrP^(sc) is that it isalmost identical to the natural form of the protein PrP^(c). The naturalfunction of this protein. is not yet understood but the remarkablystrong conservation of primary structure between homologous proteinsfrom different species suggests that it has an essential structural orfunctional role within the organism.

In spite of the almost identical form of these prions to the naturalproteins, we have deduced synthetic peptide structures comprising atleast one antigenic property, such as an epitopic site and thesesynthetic peptides may be used to produce diagnostic agents andvaccines.

The responses of the B and T cells of the immune system are notspecified by a global recognition of a whole protein but rather byrecognition of a small region of the protein surface known as epitopicsite. Such sites may be formed by a continuous section of peptide chainor may be discontinuous, where separated sections of peptide chain arebrought together at the protein surface due to folding of the chain. Oneaim in producing a synthetic peptide vaccine is to mimic the structureof a particular epitope and thereby cause a primary immune responseleading to the production of memory B cells which will secreteantibodies on subsequent exposure to the parent protein so producing agreatly enhanced response to secondary infection. A similar mechanismvia priming of the cytotoxic T cells to respond more vigorously to aparticular antigen will also occur.

However, problems exist with the application of traditional methods ofvaccine production to this disease as it is believed that the molecularstructure of the protein prion rather than nucleic acid sequence passeson infectivity in the prion. The usual method of viral vaccineproduction involves the inactivation of the virus in some way to destroyinfectivity whilst preserving epitopic sites. Such techniques as heattreatment or serial passaging of the virus through a culture are used,but these approaches would not lead to a loss of infectivity of a prionunless conditions were such as to cause protein denaturation. If theconditions are severe enough to inactivate the prion protein thendenaturation of the protein occurs and any epitopic sites are lost. Thusthere is a major problem in trying to obtain antigenic but non-infectiveprion proteins by conventional routes. It is known, for example, thatthe scrapie agent in sheep is particularly resistant to chemical orphysical inactivation (Hodgson,, J. Bio/Technology 8 990 (1990)).

In one aspect our invention provides a synthetic polypeptide having atleast one antigenic site of a prion protein. Preferably the prionprotein is of a form which only exists in nervous tissue of a mammalsuffering from spongiform encephalopathy.

We have found that prion proteins of the type mentioned above comprisesix regions of interest, labelled A to F, and two related frame shiftpeptide sequences, viz:1) a repeating section in region E havingundergone a nucleic acid coding sequence frame shift of +1 (FSa) and 2)the repeating section in region E having undergone a nucleic acid codingsequence frame shift of −1 (FSb).

With regard to region A, our invention provides a synthetic peptidesequence according to general Formula (I): (SEQ ID NO: 52)

X-(R₁-Lys-His-R₂)-Ala-Gly-Ala-Ala-Ala-R₃-Gly-Ala-Val-—Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-R₄-R₅)-Y  (I)

wherein R₁ is an amino acid residue selected from Met, Leu and Phe;

R₂ is either Met or Val;

R₃ is Ala or is absent;

R₄ and R₅ are independently an amino acid residue selected from Leu, Ileand Met; one or more residues within brackets maybe present or absentwith the proviso that if they are present they are attached to the restof the peptide in sequence; and X and Y may each independently be absentor independently be one or more additional amino acid residues.

It will be apparent for example that the residues at the N-terminal ofthe sequence may be present as “R₂”- or “His-R₂-,” or “Lys-His-R₂-” or“R₁-Lys-His-R₂-.” Similarly, the preferable residues at the C-terminalmay be present as “-Arg”, or “-Arg-Pro,” or “-Arg-Pro-R₄,” or“-Arg-Pro-R₄-R₅.”

Preferably, R₁, if present, is Met, R₃ is Ala and R₅, if present, isIle. Also, if R₂ is Met then R₄, if present, is Ile. Below are preferredsequences (Seq. I.D. No: 1 and Seq. I.D. No: 2) of formula I relating tobovine and ovine and to human prion proteins respectively:

Seq. I.D. No: 1

X-(Met-Lys-His-Val)-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Leu-Ile)-Y;and

Seq. I.D. No: 2

X-(Met-Lys-His-Met)-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Ile-Ile)-Y.

A particularly preferred sequence according to formula I is Seq. I.D.No: 51

Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-Arg-Gly-Cys.

Naturally, our invention encompasses significant sub-fragments of thesequence according to formula I above and preferred sub-fragments are:

i) X-(His-R₂-Ala-Gly)-Ala-Ala-Ala-R₃-Gly-Ala-Val-Val-(Gly-Gly-Leu-Gly)-Yand;

ii)X-(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-R₄-R₅)-Y

wherein R₂, R₃, R₄, R₅, X and Y are as defined for formula I and one ormore residues in brackets may be absent or present as in formula I.

It will be clear from the foregoing that preferred sub-fragmentsrelating to both bovines and ovines are

Seq. I.D. No: 3

i)X-(His-Val-Ala-Gly)-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-(Gly-Leu-Gly-Gly)-Y;and

Seq. I.D. No: 4

ii)X-(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Leu-Ile)-Y.

Similarly, preferred sub-fragments for humans are:

Seq. I.D. No: 5

i)X-(His-Met-Ala-Gly)-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-(Gly-Leu-Gly-Gly)-Y;and

Seq. I.D. No: 6

ii)X-(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Ile-Ile)-Y.

With regard to region B, our invention provides a synthetic peptidesequence according to general Formula II (SEQ. ID NO: 53):

X-(Ser-Ala-Met-Ser)-Arg-Pro-R₄-R₅-His-Phe-Gly-R₆-Asp—R₇-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-R₈-Arg-(Tyr-Pro-Asn-Gln)-YII (SEQ ID NO: 53)

wherein R₄ and R₅ are the same as in formula I;

R₆ is either Asn or Ser;

R₇ is either Tyr or Trp;

R₈ is an amino acid residue selected from His, Tyr and Asn;

one or more residues within brackets maybe present or absent with theproviso that if they are present they are attached to the rest of thepeptide in sequence; and

X and Y may each independently be absent or independently be one or moreadditional amino acid residues.

Preferably in a sequence according to Formula II (SEQ ID NO: 53), R₅ isIle, R₇ is Tyr and R₈ is His or Tyr. Below are preferred sequences offormula II relating to bovine, ovine and human prion proteinsrespectively:

Seq. I.D. No: 7

X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-His-Arg-(Tyr-Pro-Asn-Gln)-Y;

Seq. I.D.. No: 8

X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly-Asn-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-Tyr-Arg-(Tyr-Pro-Asn-Gln)-Y;and

Seq. I.D. No: 9

X-(Ser-Ala-Met-Ser)-Arg-Pro-Ile-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-His-Arg-(Tyr-Pro-Asn-Gln)-Y.

Particularly preferred sequences are selected from Seq. I.D. No: 42

Ser-Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Asn-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Gly-Cys;and

Seq. I.D. No: 43

Ser-Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Gly-Cys.

Again it will be apparent that our invention encompasses significantsub-fragments of the sequence according to FORMULA II and a preferredgeneral sub-fragment has the sequence:

X-(Ser-Ala-Met-Ser)-Arg-Pro-R₄-R₅-His-Phe-Gly-R₆-Asp-R₇-Glu-Asp-Arg-Tyr-Tyr-(Arg-Glu-Asn-Met)-Y

wherein R₄ to R₇, X and Y are as defined in Formula II (SEQ ID NO: 53)and one or more residues in brackets may be present or absent.Preferably, R₅ is Ile and R₇ is Tyr. It will be appreciated thatpreferred sub-fragments relating to bovines, ovines and humans arerespectively;

Seq. I.D. No: 10

X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-(Arg-Glu-Asn-Met)-Y;

Seq. I.D. No: 11

X-(Ser-Ala-Met-Ser)-Arg-Pro-Leu-Ile-His-Phe-Gly-Asn-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-(Arg-Glu-Asn-Met)-Y;and

Seq. I.D. No: 12

X-(Ser-Ala-Met-Ser)-Arg-Pro-Ile-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-(Arg-Glu-Asn-Met)-Y.

Our invention provides in respect of region C a synthetic peptidesequence according to general Formula III (SEQ ID NO: 54):

X-(Asn-Met-R₈-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-R₉-Asp—R₁₀-Tyr-R₁₁-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-YIII (SEQ ID NO: 54)

wherein R₈ is an amino acid residue selected from His, Tyr and Asn;

R₉ is Val or Met;

R₁₀ is an amino acid residue selected from Gln, Glu and Arg;

R₁₁ is Ser or Asn; one or more residues within brackets maybe present orabsent with the proviso that if they are present they are attached tothe rest of the peptide in sequence and X and Y may each independentlybe absent or independently be one or more additional amino acidresidues.

Preferably in a sequence according to Formula III (SEQ ID NO: 54), R₈ isHis or Tyr and R₁₁ is Ser. Below are preferred sequences of formula IIIrelating to bovine, ovine and human prion proteins respectively:

Seq. I.D. No: 13

X-(Asn-Met-His-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y;

Seq. I.D. No: 14

X-(Asn-Met-Tyr-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Arg-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y;and

Seq. I.D. No: 15

X-(Asn-Met-His-Arg)-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Met-Asp-Glu-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y.

Particularly preferred sequences are selected from Seq. I.D. No: 44

Asn-Met-Tyr-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Arg-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Gly-Cys;and

Seq. I.D. No: 45

Asn-Met-His-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Gly-Cys.

Significant sub-fragments of the sequence according to Formula III (SEQID NO: 54) form part of this invention and a preferred sub-fragment hasthe sequence:

X-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-R₉-Asp-R₁₀-Tyr-R₁₁-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y.

Preferred sub-fragments relating to bovines, ovines and humans arerespectively:

Seq. I.D. No: 16

X-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y;

Seq. I.D. No: 17

X-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Arg-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y;and

Seq. I.D. No: 18

X-(Arg-Tyr-Pro-Asn)-Gln-Val-Tyr-Tyr-Arg-Pro-Met-Asp-Glu-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-(Asp-Cys-Val-Asn)-Y.

In respect of region D, our invention provides a synthetic peptidesequence according to general Formula IV (SEQ ID NO: 55):

X-(Tyr-Tyr-R₁₂-R₁₃-Arg)-R₁₄-R₁₅-Ser-R₁₆-R₁₇-R₁₈—Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-Leu-Leu-Ile-Ser-Phe-Leu-ILe-phe-Leu-R₁₉-Val-Gly-YIV (SEQ ID NO: 55)

wherein R₁₂ is Asp or Gln;

R₁₃ is Gly or absent;

R₁₄ is Gly or Arg;

R₁₅ is Ala or Ser;

R₁₆ is Ser or absent;

R₁₇ is an amino acid residue selected from Ala, Thr, Met and Val;

R₁₈ is Val or Ile;

R₁₉ is Ile or Met; one or more residues within brackets may be presentor absent with the proviso that if they are present they are attached tothe rest of the peptide in sequence and X and Y may each independentlybe absent or independently be one or more additional amino acidresidues.

Preferably in a sequence according to Formula IV (SEQ ID NO: 55) R₁₂ isGln, R₁₃ is absent, R₁₄ is Gly, R₁₆ is absent, R₁₇ is Val or Met and R₁₉is Ile.

Preferred sequences of Formula IV (SEQ ID NO: 55) relating to bovine andovine and to human prion proteins respectively are given below:

Seq. I.D. No: 19

X-(Tyr-Tyr-Gln-Arg)-Gly-Ala-Ser-Val-Ile-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-Leu-Leu-Ile-Ser-Phe-Leu-Ile-Phe-Leu-Ile-Val-Gly-Y;and

Seq. I.D. No: 20

X-(Tyr-Tyr-Gln-Arg)-Gly-Ser-Ser-Met-Val-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-Leu-Leu-Ile-Ser-Phe-Leu-Ile-Phe-Leu-Ile-Val-Gly-Y.

Clearly, it will be recognised that the present invention includes withits ambit significant subfragments of the sequence according to FormulaIV (SEQ ID NO: 55) and a preferred general sub-fragment has thesequence:

X-(-R₁₄-R₁₅-Ser-R₁₆-R₁₇)-R₁₈-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-(Leu-Leu-Ile-Ser)-y

Wherein R₁₄ to R₁₈, X and Y are as defined in formula IV and one or moreresidues within brackets may be present or absent as in formula IV.

It is preferred that in a sub-fragment as given above, R₁₄ is Gly, R₁₆is absent and R₁₇ is Val or Met. Below are preferred sub-fragmentsrelating to bovines and ovines and to humans respectively:

Seq. I.D. No: 21

X-(Gly-Ala-Ser-Val)Ile-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-(Leu-Leu-Ile-Ser)-Y; and

Seq. I.D. No: 22

X-(Gly-Ser-Ser-Met)-Val-Leu-Phe-Ser-Ser-Pro-Pro-Val-Ile-(Leu-Leu-Ile-Ser)-Y.

Our invention provides in respect of Region E three syntheticpolypeptide sequences according to general formulae VA (SEQ ID NO: 56),Vb (SEQ ID NO: 57), and Vc (SEQ ID NO: 58):

X-(Pro-Gly-Gly-R₂₀)-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly—Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-(Gly-R₂₁-R₂₂-Trp)-YVA (SEQ ID NO: 56)

X-(Gly-Gly-R₂₁-R₂₂-Trp)-Gly-Gln-Pro-His-Gly-Gly-Gly-R₂₃-Trp(Gly-Gln-Pro-His)-YVb (SEQ ID NO: 57) and

X-(Gly-Gly-GIy-Trp)-Gly-Gln-Gly-Gly-R₂₄-R₂₅-His-R₂₆-Gln-Trp-Asn-Lys-Pro-R₂₇—Lys-Pro-Lys-Thr-R₂₈-R₂₉-Lys(-His-R₃₀-Ala-Gly)-YVC (SEQ ID NO: 58).

Wherein R₂₀, R₂₁, R₂₃ and R₂₄ are each independently either Gly orabsent;

R₂₂ either Gly or Thr;

R₂₅ is either Thr or Ser;

R₂₆ is an amino acid residue selected from Gly, Ser and Asn;

R₂₇ and R₂₈ are each independently either Asn or Ser;

R₂₉ is an amino acid residue selected from Met, Leu and Phe;

R₃₀ is either Val or Met; one or more residues within brackets maybepresent or absent with the proviso that if they are present they areattached to the rest of the peptide in sequence; and X and Y may eachindependently be absent or independently be one or more additional aminoacid residues.

With regard to formulae Va to Vc above, it is preferred that R₂₂ is Gly,R₂₃ is absent, R₂₆ is Gly or Ser, R₂₇ is Ser, R₂₈ is Asn and R₂₉ is Met.

Preferred bovine sequences of prion proteins according to formulae Va toVc are given below:

Seq. I.D. No: 23

X-(Pro-Gly-Gly-Gly)-Trp-Asn-Thr-Gly-Gly-Ser-Atg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-(Gly-Gly-Gly-Trp)-Y;

Seq. I.D. No: 24

X-(Gly-Gly-Gly-Trp)-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-(Gly-Gln-Pro-His)-Y;and

Seq. I.D. No: 25

X-(Gly-Gly-Gly-Trp)-Gly-Gln-Gly-Gly-Thr-His-Gly-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn-Met-Lys(-His-Val-Ala-Gly)-Y.

Preferred sequences of formulae Va to Vc relating to ovine prionproteins are as follows:

Seq. I.D. No: 26

X-(Pro-Gly-Gly-Gly)-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-(Gly-Gly-Gly-Trp)-Y;

Seq. I.D. No: 27

X-(Gly-Gly-Gly-Trp)-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-(Gly-Gln-Pro-His)-Y;and

Seq. I.D. No: 28

X-(Gly-Gly-Gly-Trp)-Gly-Gln-Gly-Gly-Ser-His-Ser-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn-Met-Lys(-His-Val-Ala-Gly)-Y.

Preferred sequences of Formulae Va to Vc relating to human prionproteins are as follows:

Seq. I.D. No: 29

X-Pro-Gly-Gly-Gly-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-(Gly-Gly-Gly-Trp)-Y;

Seq. I.D. No: 30

X-(Gly-Gly-Gly-Trp)-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-(Gly-Gln-Pro-His)-Y;and

Seq. I.D. No: 31

X-(Gly-Gly-Gly-Trp)-Gly-Gln-Gly-Gly-Gly-Thr-His-Ser-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn-Met-Lys(-His-Met-Ala-Gly)-Y.

Particularly preferred sequences of Formulae Va to Vc consist of:

Seq. I.D. No: 49

Gly-Gly-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-Gly-Gly-Cys;

Seq. I.D. No: 46

Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Cys;and

Seq. I.D. No: 47

Gly-Gln-Gly-Gly-Ser-His-Ser-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn-Met-Lys-His-Val-Gly-Cys.

We have noted that in the nucleic acid sequence corresponding to regionE, it is possible for the repeating sequence of formula Vb(SEQ ID NO:57) to have undergone a frame shift of either +1 or −1. Such frameshifts give rise to altered sequences in region E of the prion proteinand our invention provides a synthetic polypeptide having a sequencewherein a repeat in region E has undergone a −1 frame shift as given inFormula VI(SEQ ID NO: 59)

X-(R₃₁-R₃₂-Trp-R₃₃)-Trp-Leu-Gly-R₃₄-R₃₅-R₃₆-Trp-R₃₇ (Trp-Leu-Gly-R₃₈)-Y(VI)(SEQ ID NO: 59)

Wherein R₃₁ and R₃₅, are each independently either Ala or Thr; R₃₂ andR₃₆ are each independently an amino acid residue selected from Ser, Proand Thr; R₃₃ and R₃₇ are each independently either Trp or Arg; R₃₄ andR₃₈ are each independently an amino acid residue selected from Ala, Ser,Pro and Thr; one or more residues within brackets maybe present orabsent with the proviso that if they are present they are attached tothe rest of the peptide in sequence; and X and Y may each independentlybe absent or independently be one or more, additional amino acidresidues.

With regard to −1 frame shifts in respect of region E in bovines, it ispreferred that R₃₁ is Ala, R₃₂, R₃₄, R₃₆ and R₃₈ are each independentlyeither Ser or Pro, R₃₃ and R₃₇ are Arg and R₃₅ is Ala.

It should be noted that preferred sequences for −1 frame shifts inregion E of ovines differ in some respects to those given for bovinesand in a preferred ovine sequence R₃₁, R₃₂, R₃₃, R₃₅, R₃₆ and R₃₇correspond to the definitions given for formula VI above; and R₃₄ andR₃₈ are each independently selected from Ser, Pro and Thr.

In a preferred human sequence according to formula VI R₃₁, R₃₄, R₃₅ andR₃₈ are each Ala, R₃₂ and R₃₆ are each independently either Ser or Proand R₃₃ and R₃₇ are both Trp.

As mentioned previously, the frame shift may be +1 in the repeat portionof region E and this gives rise to different amino acid sequences.Accordingly, our invention provides a synthetic polypeptide according toFormula VII(SEQ ID NO: 60) below which relates to a +1 frame shift inthe repeat of region E:

X-(R₃₉-R₄₀-Met-R₄₁)-Val-Ala-Gly-R₄₂-R₄₃-R₄₄-Met-R₄₅-(Val-Ala-Gly-R₆)-YVII(SEQ ID NO: 60)

Wherein R₃₉ and R₄₃ are each independently either Ser or Asn; R₄₀ andR₄₄ are each independently an amino acid residue selected from Pro, Leuand His, R₄₁ and R₄₅ are each independently Val or Glu; R₄₂ and R₄₆ areeach independently selected from Val, Ala, Asp and Gly; one or moreresidues within brackets maybe present or absent with the proviso thatif they are present they are attached to the rest of the peptide insequence; and X and Y may each independently be absent or independentlybe one or more, additional amino acid residues.

A preferred bovine sequence according to Formula VII(SEQ ID NO: 60)comprises R₃₉ and R₄₃ each being Ser, R₄₂ and R₄₆ each beingindependently either Val or Ala and R₄₄ being either Pro or Leu; withthe other R groups being as defined in Formula VII(SEQ ID NO: 60).

A preferred sequence according to formula VII relating to ovines is thesame as given in general formula VII except R₄₂ and R₄₆ are eachindependently selected from Val, Ala and Asp.

With regard to a preferred human sequence according to formula VII, R₃₉and R₄₃ are Ser, R₄₀ and R₄₄ are each independently Pro or Leu, R₄₁ andR₄₅ are Val and R₄₂ and R₄₆ are each independently either Asp or Gly

Our invention also provides a synthetic peptide sequence relating toregion F and having either the general Formula VIIIA(SEQ ID NO: 61) andVIIIB(SEQ ID NO: 62).

X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-R₄₇-Lys-R₄₈-His-Thr-Val—R₄₉-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Thr-Asp-R₅₀-Lys)-Y(VIIIA(SEQ ID NO: 61)

X-(Met-Cys-R₅₁-Thr)-Gln-Tyr-R₅₂-R₅₃-Glu-Ser-Gln-Ala—Tyr-Tyr-R₅₄-R₅₅-Arg-(R₅₆-R₅₇-Ser-R₅₈-R₅₉)-Y(VIIIB(SEQ ID NO: 62)

Wherein R₄₇ is either Ile or Val;

R₄₈ and R₅₂ are each independently either Gln or Glu;

R₄₉ is either Val or Thr;

R₅₀ is either Val or Ile;

R₅₁ is an amino acid residue selected from Ile, Thr and Val;

R₅₂ is Gln or Glu;

R₅₃ is either Arg or Lys;

R₅₄ is either Asp or Gln;

R₅₅ is Gly or is absent;

R₅₆ is either Gly or Arg;

R₅₇ is either Ala or Ser;

R₅₈ is Ser or absent;

R₅₉ is an amino acid residue selected from Ala, Thr, Met and Val;

one or more residues within brackets maybe present or absent with theproviso that if they are present they are attached to the rest of thepeptide in sequence; and X and Y may each independently be absent orindependently be one or more, e.g,. 3, additional amino acid residues.

It is preferred in Formula VIIIA(SEQ ID NO: 61) that R₄₉ is Thr and inFormula VIIIB(SEQ ID NO: 62) that R₅₁ is Ile, R₅₃ is Arg, R₅₄ is Gln,R₅₅ is absent, R₅₆ is Gly, R₅₇ is Ala and R₅₈ is absent.

Most preferred bovine, ovine and human sequences according to FormulaeVIIIA(SEQ ID NO: 61) and VIIIB(SEQ ID NO: 62) are given below in order:

Seq. I.D. No: 32

X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-Val-Lys-Glu-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Thr-Asp-Ile-Lys)-Ybovine (VIIIa), and

Seq. I.D. No: 33

X-(Met-Cys-Ile-Thr)-Gln-Tyr-Gln-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg-(Gly-Ala-Ser-Val)-Ybovine (VIIIb);

Seq. I.D. No: 34

X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-Val-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Thr-Asp-Ile-Lys)-Yovine (VIIIa), and

Seq. I.D. No: 35

X-(Met-Cys-Ile-Thr)-Gln-Tyr-Gln-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg-(Gly-Ala-Ser-Val)-Yovine (VIIIb);

Seq. I.D. No: 36

X-(Asn-Phe-Val-His)-Asp-Cys-Val-Asn-Ile-Thr-Ile-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-(Thr-Asp-Val-Lys)-Yhuman (VIIIa), and

Seq. I.D. No: 37

X-(Met-Cys-Ile-Thr)-Gln-Tyr-Glu-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg-(Gly-Ser-Ser-Met)-Yhuman (VIIIb)

Particularly preferred sequences according to formula VIIIa and VIIIbare selected from

Seq. I.D. No: 50

Val-Asn-Ile-Thr-Val-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-Gly-Cys;and

Seq. I.D. No: 48

Cys-Ile-Thr-Gln-Tyr-Gln-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg.

Synthetic polypeptides according to any one of Formulae I to VIIIb abovewithout X and Y being present will of course be useful, for example, inthe production of antibodies. However, when X or Y are present they maybe any length but preferably less than 20 amino acids, more preferablyless than 10, eg. 3 to 6. It will of course be appreciated that asequence according to any one of Formulae I to VIIIb may constitute aprotein with X and Y being major portions of the protein with theantigenic sequence being for example, part of an exposed loop on aglobular protein.

It is preferred that if X or Y are present they are relatively shortsequences, typically 1 to 3 residues long. In most instances X ispreferably absent and Y is 1 or 2 residues long, e.g. -Cys or -Gly-Cys.

All the sequences herein are stated -using the standard I.U.P.A.C.three-letter-code abbreviations for amino acid residues defined asfollows: Gly-Glycine, Ala-Alanine, Val-Valine, Leu-Leucine,Ile-Isoleucine, Ser-Serine, Thr-Threonine, Asp-Aspartic acid,Glu-Glutamic acid, Asn-Asparagine, Gln-Glutamine, Lys-Lysine,His-Histidine, Arg-Arginine, Phe-Phenylalanine, Tyr-Tyrosine,Trp-Tryptophan, Cys-Cysteine, Met-Methionine and Pro-Proline.

Polypeptides according to the invention may be used to raise antibodieswhich will cross-react with prion proteins produced in a wide range oforganisms. Our analyses have shown that since the conformational,topographic and electrostatic properties of polypeptides according tothe invention are such that they are highly likely to elicit theproduction of antibodies which will cross-react with prion proteins fromseveral or many organisms, further advantages may arise from combiningseveral variant polypeptides in a larger polypeptide. Such a polypeptidemay have the general Formula (IX):

[L_(a)−F]_(m)[L_(b)−G]_(n)−L_(c)  (IX)

wherein F and G may each independently be a polypeptide or sub-fragmentaccording to any one of Formulae I to VIIIb, L is a linking sequence, a,b and c are each independently 0 or 1 and m and n are each positivenumbers e.g. between 1 and 10 inclusive. L is preferably a short,conformationally flexible section of polypeptide chain such as, forexample and without limit (Seq. I.D. No: 38) Gly-Gly-Gly-Gly-Gly, (Seq.I.D. No: 39) Gly-Pro-Gly-Pro-Gly-Pro or (Seq. I.D. No: 40)Gly-Ser-Ala-Gly-Ser-Gly-Ala. It should be clear that each repeat mayoptionally have a different variant of a polypeptide according to theinvention.

It should be noted certain of the C-teminals correspond to N-terminals,particularly Formula VA(SEQ ID NO: 66) to formula Vb, formula VC(SEQ IDNO: 58) to formula I, formula I to Formula II, Formula II(SEQ ID NO: 53)to formula III, formula III to formula VIIIa and formula VIIIb toformula IV. Advantage may be taken to this correspondence when producinglarger polypeptides according to formula IX. Linking sequences togetherwith respective X and Y moieties may be omitted and residues in bracketsmay be selected so that either the regions of correspondence areduplicated or some or all of the duplicated residues are omitted. In thelatter case it will be seen that the C-terminal of one polypeptidemerges with the N-terminal of the other polypeptide.

Polyvalent determinant analogues as defined by Formula IX may be eitherwhat is referred to as pseudohomopolyvalent, wherein variants ofessentially the same determinant analogue are repeated in a singlepolypeptide chain and/or heteropolyvalent, wherein distinct determinantsare included in a single polypeptide. In addition, simple homopolvvalentpolypeptide immunogens, which contain multiple copies of the samevariant of one of the determinant analogues according to any one offormulae I to VIIIb, would also be expected to be effective, and arealso included within the scope of the present invention.

It is to be understood that any antigenically significant subfragmentsand/or antigenically significant variants of the above-identifiedpolypeptide sequences which retain the general form and function of theparent polypeptide are included within the scope of this invention. Inparticular, the substitution of any of the specific residues by residueshaving comparable conformational and/or physical properties, includingsubstitution by rare (but naturally occurring, e.g. D-stereoisomers) orsynthetic amino acid analogues, is included. For example, substitutionof a residue by another in the same Set, as defined below, is includedwithin the ambit of the invention; Set 1—Ala, Val, Leu, Ile, Phe, Tyr,Trp and Met; Set 2—Ser, Thr, Asn and Gln; Set 3—Asp and Glu; Set 4—Lys,His and Arg; Set 5—Asn and Asp; Set 6—Glu and Gln; Set 7—Gly, Ala, Pro,Ser and Thr. D-stereoisomers of all amino acid types, may besubstituted, for example, D-Phe, D-Tyr and D-Trp.

In preferred embodiments of The invention, X and Y if present mayindependently include one or more segments of protein sequence with theability to act as a T-cell epitope. For example, segments of amino acidsequence of the general formula 1-2-3-4, where 1 is Gly or a chargedamino acid (e.g. Lys, His, Arg, Asp or Glu), 2 is a hydrophobic aminoacid (e.g. Ile, Leu, Val, Met, Tyr, Phe, Trp, Ala), 3 is-either ahydrophobic amino acid (as defined above) or an uncharged polar aminoacid (e.g. Asn, Ser, Thr, Pro, Gln, Gly), and 4 is a polar amino acid(e.g. Lys, Arg, His, Glu, Asp, Asn, Gln, Ser, Thr, Pro), appear to actas T-cell epitopes in at least some instances (Rothbard, J. B. & Taylor,W. R. (1988). A sequence pattern in common to T-cell epitopes. The EMBOJournal 7(1): 93-100). Similarly segments can be of the sequence1′-2′-3′-4′-5′, wherein 1′ is equivalent to 1 as defined earlier, 2′ to2, 3′ and 4′ to 3, and 5′ to 4 (ibid). Both forms are included withinthe scope of the present invention and one or more T-cell epitopes(preferably less than five) which may be of the type defined above ormay be of other structure and which may be separated by spacer segmentsof any length or composition, preferably less than five amino acidresidues in length and comprising for example residues selected fromGly, Ala, Pro, Asn, Thr, Ser or polyfunctional linkers such as non-αamino acids. It is possible for a C- or N-terminal linker to represent acomplete protein, thus obviating the possible need for conjugation to acarrier protein.

Also included within the scope of this invention are derivatives of thepolypeptides according to any one Formulae I to VIIIb in which X or Yare or include a “retro-inverso” amino acid, i.e. a bifunctional aminehaving a functional group corresponding to an amino acid. For example ananalogue according to the invention and containing a retro-inverso aminoacid may have the formula:

where R is any functional group, e.g. a glycine side chain, and A1 andA2 are preferably each a copy of one of the analogues defined herein(but not necessarily the same) attached by its N- or C-terminal end.T-cell epitopes may optionally be included as discussed earlier.

Retro-inverso modification of peptides involves the reversal of one ormore peptide bonds to create analogues more resistant than the originalmolecule to enzymatic degradation and offer one convenient route to thegeneration of branched immunogens which contain a high concentration ofepitope for a medium to large immunogen. The use of these compounds inlarge-scale solution synthesis of retro-inverso analogues of short-chainbiologically active peptides has great potential.

Peptides according to the invention may be synthesised by standardpeptide synthesis techniques, for example using either standard9-fluorenyl-methoxycarbonyl (F-Moc) chemistry (see, for example,Atherton, E. and Sheppard, R. C. (1985) J. Chem. Soc. Chem. Comm. 165)or standard butyloxycarbonate (T-Boc) chemistry although it is notedthat, more recently, the fluorenylmethoxycarbonyl (Fmoc)/tert-butylsystem, developed by Sheppard et al has found increasingly wideapplication (Sheppard, R. C. 1986 Science Tools, The LKB Journal 33, 9).The correctness of the structure and the level of purity, which willnormally be in excess of 85%, should be carefully checked, andparticular attention be given to the correctness of internal disulphidebridging arrangements when present. Various chromatographic analyses,including high performance liquid chromatography, and spectrographicanalyses, including Raman spectroscopy, may for example be employed forthis purpose.

It is to be understood that the polypeptides according to the inventionmay be synthesised by any conventional method, either directly usingmanual or automated peptide synthesis techniques as mentioned above, orindirectly by RNA or DNA synthesis and conventional techniques ofmolecular biology and genetic engineering. Such techniques may be usedto produce hybrid proteins containing one or more of the polypeptidesinserted into another polypeptide sequence.

Another aspect of the present invention therefore provides a DNAmolecule coding for at least one synthetic polypeptide according to theinvention, preferably incorporated into a suitable expression vectorreplicable in microorganisms or in mammalian cells. The DNA may also bepart of the DNA sequence for a longer product e.g. the polypeptides maybe expressed as parts of other proteins into which they have beeninserted by genetic engineering. One practical guide to such techniquesis “Molecular cloning: a laboratory manual” by Sambrook, J., Fritsch, E.F. and Maniatis, T. (2 nd Edition, 1989).

It should be noted that analogues incorporating retro-inverso amino acidderivatives cannot be made directly using a recombinant DNA system.However, the basic analogues can, and they can then be purified andchemically linked to the retro-inverso amino-acids using standardpeptide/organic chemistry. A practical and convenient novel procedurefor the solid-phase synthesis on polyamide-type resin of retro-inversopeptides has been described recently [Gazerro, H., Pinori, M. & Verdini,A. S. (1990). A new general procedure for the solid-phase synthesis ofretro-inverso peptides. In “Innovation and Perspectives in Solid phaseSynthesis” Ed. Roger Epton. SPCC (UK) Ltd, Birmingham, UK].

The polypeptides are optionally linked to a carrier molecule, eitherthrough chemical groups within the polypeptides themselves or throughadditional amino acids added at either the C- or N-terminus, and whichmay be separated from the polypeptides themselves or surrounded by oneor more additional amino acids, in order to render them optimal fortheir immunological function. Many linkages are suitable and include forexample use of the side chains of Tyr, Cys and Lys residues. Suitablecarriers include, for example, purified protein derivative of tuberculin(PPD), tetanus toxoid (TT), cholera toxin and its B subunit, ovalbumin,bovine serum albumin (BSA), soybean trypsin inhibitor (STI), muramyldipeptide (MDP) and analogues thereof, diphtheria toxoid (DPT), keyholelimpet haemocyanin (KLH) and Braun's lipoprotein although other suitablecarriers will be apparent to the skilled person. For example, multiple atigen peptides may be used such as those comprising a polylysyl core,e.g. heptalysyl, bearing reactive amine termini. Polypeptide antigensaccording to the invention may be reacted with, or synthesised on, theamino termini and different polypeptide antigens may be reacted with thesame core or carrier. When using PPD as a carrier for polypeptidesaccording to the invention, a higher titre of antibodies is achieved ifthe recipient of the polypeptide-PPD conjugate is already tuberculinsensitive, e.g. by virtue of earlier BCG vaccination. In the case of ahuman vaccine it is worth noting that in the UK and many other countriesthe population is routinely offered BCG vaccination and is thereforelargely PPD-sensitive. Hence PPD is expected to be a preferred carrierfor use in such countries.

The mode of coupling the polypeptide to the carrier will depend on thenature of the materials to be coupled. For example, a lysine residue inthe carrier may be coupled to a C-terminal or other cysteine residue ina polypeptide by treatment with N-γ-maleimidobutyryloxy-succinimide(Kitagawa, T. & Ackawa, T. (1976) J. Biochem. 79, 233). Alternatively, alysine residue in the carrier may be conjugated to a glutamic oraspactic acid residue in the peptide using isobutylchloroformate(Thorell, J. I. De Larson, S. M. (1978) Radioimmunoassay and relatedtechniques: Methodology and clinical applications, p.288). Othercoupling reactions and reagents have been described in the literature.

The polypeptides, either alone or linked to a carrier molecule, may beadministered by any route (eg parenteral, nasal, oral, rectal,intra-vaginal), with or without the use of conventional adjuvants (suchas aluminium hydroxide or Freund's complete or incomplete adjuvants)and/or other immunopotentiating agents. The invention also includesformulation of polypeptides according to the invention in slow-releaseforms, such as a sub-dermal implant or depot comprising, for example,liposomes (Allison, A. C. & Gregoriadis, G. (1974) Nature (London) 252,252) or biodegradable microcapsules manufactured from co-polymers oflactic acid and glycolic acids (Gresser, J. D. and Sanderson, J. E.(1984) in “Biopolymer Controlled Release Systems” pp 127-138, Ed. D. L.Wise).

Polypeptides according to the invention may be used either alone orlinked to an appropriate carrier, as:

(a) As ligands in assays of, for example, serum from patients oranimals;

(b) Peptide vaccines, for use in prophylaxis;

(c) As quality control agents in testing, for example, binding levels ofantibodies raised against the polypeptides;

(d) As antigenic agents for the generation of monoclonal or polyclonalantibodies by immunisation of an appropriate animal, such antibodiesbeing of use for (i) the scientific study of prion proteins, (ii) asdiagnostic agents, e.g. as part of immunohistochemical reagents, (iii)for the passive immunisation of animals or patients, either as atreatment for encephalophathies or in combination with other agents,(iv) as a means of targeting other agents to regions comprising prionproteins, such agents either being linked covalently or otherwiseassociated, e.g. as in liposomes containing such agents andincorporating antibodies raised against any of the antigenicpolypeptides and (v) for use as immunogens to raise anti-idiotypeantibodies; such anti-idiotype antibodies also form part of thisinvention. The invention further provides for cometically engineeredforms or sub-components, especially V_(H) regions, of antibodies raisedagainst the polypeptides, and of ovinised, bovinised, or humanised formsof antibodies initially raised against the polypeptides in otheranimals, using techniques described in the literature; and

(e) The treatment of encephalopathies, either by displacing the bindingof prion proteins to human or animal cells or by disturbing thethree-dimensional organisation of the protein in vivo; as well as aidingthe scientific study of prion proteins in vitro.

In respect of detection and diagnosis, of prion proteins or antibodiesagainst prion proteins, the skilled person will be aware of a variety ofimmunoassay techniques known in the art, inter alia, sandwich assay,competitive and non-competitive assays and the use of direct andindirect labelling.

A further aspect of the invention provides a kit for detecting prionproteins or antibodies against prion proteins which comprises at leastone synthetic polypeptide according to the invention. The

The preparation of polyclonal or monoclonal antibodies, humanised formsof such antibodies (see, for example, Thompson K. M. et al (1986)Immunology 58, 157-160), single domain antibodies (see, for example,Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. and Winter, G.(1989) Nature 341, 544-546), and antibodies which might cross theblood-brain barrier, which bind specifically to a synthetic polypeptideaccording to the present invention, may be carried out by conventionalmeans and such antibodies are considered to form part of this invention.Antibodies according to the invention are, inter alia, of use in amethod of diagnosing mammalian encephalopathies which comprisesincubating a sample of tissue or body fluid of mammal with an amount ofantibody as described herein and determining whether, and if desired theextent to which and/or rate at which, cross-reaction between said sampleand said antibody occurs. Diagnostic kits which contain at least one ofsaid antibodies also form part of this invention.

A further aspect of the invention provides. synthetic polypeptides foruse in therapy or prophylaxis of mammalian encephalopathies and/orstimulating the mammalian immune system and/or blocking the cellularbinding or aggregation of the prion proteins and for the preparation ofmedicaments suitable for such uses. Also included are pharmaceuticalcompositions containing, as active ingredient, at least one polypeptideor polypeptide-carrier conjugate as described herein in association withone or more pharmaceutically acceptable adjuvants, carriers and/orexcipients. The compositions may be formulated for oral, rectal, nasalor especially parenteral administration (including intra-CNSadministration).

The invention further provides a method of therapy or prophylaxis ofmammalian encephalopathies and/or of stimulating the mammalian immunesystem and/or of blocking the cellular binding or aggregation of theprion proteins, which comprises administering an amount of a polypeptideas hereinbefore defined, either in isolation or in combination withother agents for the treatment of encephalopathies.

Discrimination between natural PrP^(c) and PrP^(sc) is highly desiredsince PrP^(c) is found in normal subjects and both PrP^(c) and PrP^(sc)are found in a diseased subject. We have found that peptide sequencesaccording to the invention, preferably those relating to regions A, Band C, and significant sub-fragments thereof may be used to discriminatebetween natural PrP^(c) and infective PrP^(sc). Also, antibodies raisedagainst these peptide sequences and sub-fragments and the nucleotidesequences which code for such peptide sequences and sub-fragments mayalso be used to discriminate between PrP^(c) and PrP^(sc). Accordingly,the invention provides a method of discriminating between PrP^(c) andPrP^(sc) in which a sample is contacted with a substance selected frompeptide sequences according to the invention, preferably those reletingto regions A, B and C, and significant sub-fragments thereof, antibodiesraised against said sequences and sub-fragments and the presence orabsence of PrP^(sc) is determined.

In some instances discrimination may be enhanced by pretreatment of thesample, for example by pre-digestion with enzymes e.g. proteinase K, ordenaturation by strong alkali e.g. 6M guanidine hydrochloride or by acombination of such treatments.

It will be preferable to use the peptide sequences, antibodies andnucleotide sequences which relate to the specific subject under test,e.g. bovine sequences and antibodies for cattle, ovine sequences andantibodies for sheep.

It may be advantageous to immunise with a cocktail containing (i) agiven analogue conjugated to more than one type of carrier molecule,and/or (ii) more than one kind of analogue conjugated to the samecarrier molecule. Moreover, any of the peptide analogues, theirconjugates, and cocktails thereof may be administered in any suitableadjuvant or delivery system, and more than one adjuvant or deliverysystem may be combined to form a so-called “super-cocktail”. Preferredadjuvants and delivery systems include aluminium hydroxide (alum),liposomes, micelles, niosomes, ISCOMS, Brauns lipoprotein and whole-cellor components of microbial animal vaccines.

EXAMPLE 1

A preferred bovine form of formula II (Seq. I.D. No: 41)Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Arg-Glu-Asn-Met-His-Arg-Gly-Cys(related to seq. I.D. No: 7) in which the C-terminal Y extension isGly-Cys according to the invention is synthesised using standardsolid-phase Fmoc methodologies. The-peptide is cleaved from the resin inthe presence of trifluroacetic acid and subsequent purification isachieved by gel filtration, ion exhange chromatography and reverse phasehigh performance liquid chromatography. The peptide is conjugated to avariety of carriers by MBS (m-Maleimido-benzoyl-N-hydroxy succinimideester), a well-known hetero-bifunctional reagent.

Examples of carriers include KLH, BSA and TT which have been shown toprovide necessary immunopotentiating properties to B cell epitopes.

The peptide carrier conjugates are emulsified in Freund's CompleteAdjuvant and are administered intramuscularly to mice. Subsequentbooster injections are given in Freund's Incomplete Adjuvant.

The ensuing serum antibody response is monitored throughout theimmunisation schedule by enzyme immunoassay (ELISA) using immobilisedantigen (formula II), coupled to BSA, the serum sample under test, andan enzyme-labelled anti-mouse antibody.

In this example, use of carriers, adjuvants and delivery systems andbooster injections are effected in order to determine an optimalprotocol for producing Anti-formula II antibodies.

EXAMPLE 2

Antibodies to formula II are used as diagnostic reagents for assayingthe presence of prion protein in serum, in “cell carriers” in serum andin tissue biopsies of injected animal species.

A direct enzyme immunoassay (ELISA) can detect the presence of extractedprion protein by its immobilisation onto a solid substrate. Reaction ofmouse antisera raised to formula II with native prion protein isdetected with an enzyme-labelled anti-mouse antiserum. The reaction isquantified by addition of a suitable substrate and reading the opticaldensity of the colour produced.

Furthermore, immunohistochemical diagnosis of prion proteins in tissuebiopsies can be performed by reacting Anti-formula II antibodies withparaffin wax embedded or frozen tissue. Reactions can be detected usinga standard indirect immunoperoxidase technique.

EXAMPLE 3 MATERIALS AND METHODS

Peptide Synthesis

The following peptides were synthesised using standard solid-phase Fmocmethodologies.

Peptide II: (Seq. I.D. No: 42)

Ser-Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Asn-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Gly-Cys

(A preferred ovine sub-fraqment of formula II).

Peptide BII: (Seq. I.D. No: 43)

Ser-Ala-Met-Ser-Arg-Pro-Leu-Ile-His-Phe-Gly-Ser-Asp-Tyr-Glu-Asp-Arg-Tyr-Tyr-Gly-Cys

(A preferred bovine sub-fragment of formula II).

Peptide III: (Seq. I.D. No: 44)

Asn-Met-Tyr-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Arg-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Gly-Cys

(A preferred ovine sequence of formula III (p8, ln 30-32).

Peptide BIII: (Seq. I.D. No: 45)

Asn-Met-His-Arg-Tyr-Pro-Asn-Gln-Val-Tyr-Tyr-Arg-Pro-Val-Asp-Gln-Tyr-Ser-Asn-Gln-Asn-Asn-Phe-Val-His-Gly-Cys

(A preferred bovine sequence of formula III (p8, ln 26-28).

Peptide Vb: (Seq. I.D No: 46)

Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Gln-Pro-His-Gly-Gly-Gly-Trp-Gly-Cys

(A preferred ovine/bovine sequence of formula Vb).

Peptide Vc: (Seq. I.D. No: 47)

Gly-Gln-Gly-Gly-Ser-His-Ser-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn-Met-Lys-His-Val-Gly-Cys

(A preferred ovine sequence of formula Vc).

Peptide VIIIb: (Seq. I.D. No: 48)

Cys-Ile-Thr-Gln-Tyr-Gln-Arg-Glu-Ser-Gln-Ala-Tyr-Tyr-Gln-Arg

(A preferred ovine/bovine sequence of formula VIIIb).

Peptide Va: (Seq. I.D. No: 49)

Gly-Gly-Trp-Asn-Thr-Gly-Gly-Ser-Arg-Tyr-Pro-Gly-Gln-Gly-Ser-Pro-Gly-Gly-Asn-Arg-Tyr-Pro-Pro-Gln-Gly-Gly-Gly-Cys

Peptide VIIIa: (Seq. I.D. No: 50)

Val-Asn-Ile-Thr-Val-Lys-Gln-His-Thr-Val-Thr-Thr-Thr-Thr-Lys-Gly-Glu-Asn-Phe-Thr-Glu-Gly-Cys

(A preferred ovine sequence of formula VIIIa).

Peptide I: (Seq. I.D. No: 51)

Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-Arg-Gly-Cys.

Peptides I, II, BII, III, BIII, Va, Vb, Vc and VIIIa were synthesisedwith the C-terminal extension according to the invention. The peptideswere cleaved from the resin in the presence of trifluoroacetic acid andsubsequent purification was achieved by reverse phase high performanceliquid chromatography. All peptides had a purity of 85% or more.

Conjugation of Peptides to Ovalbumin

Peptides were conjugated through their C-terminal (peptides II, BII,III, BIII, Vb and Vc) or N-terminal (peptide VIIIb) Cys residues.Peptides were dissolved in dimethyl sulphoxide (DMSO) to a concentrationof 10 mg/ml. Preactivated ovalbumin (Pierce, Imject Kit) was resuspendedin 1 ml of distilled water, and equal volumes of preactivated ovalbuminand peptide were mixed and the solution allowed to stand at roomtemperature for 3 hours. The conjugate was dialysed overnight againstphosphate buffered saline (PBS) to remove the DMSO and unconjugatedpeptide.

The extent of conjugation was determined by measuring the free-thiolcontent using an Ellman's assay and by monitoring the increase in themolecular mass of the conjugate by SDS-PAGE (sodium dodecylsulphate-polyacrylamide gel electrophoresis).

Generation of Rabbit Antisera.

Antiserum was raised against each of the peptide conjugates in twofemale New Zealand White rabbits. Each rabbit received an amount ofconjugate equivalent to 40 μg of peptide for both the primaryinoculation and the boosters. Rabbits were injected as follows:

Day 0: Conjugate in Freund's Complete Adjuvant (1:1, v/v)intramuscularly.

Day 21: Conjugate in Freund's Incomplete Adjuvant (1:1, v/v)intramuscularly.

Day 31: Conjugate on its own intraperitoneally.

Animals were bled on day 41, and the sera assayed for anti-peptideantibody by ELISA (using free peptide as the coating antigen). The serawere also used in immunoblot and dot blot assays to see if they couldrecognise proteins from the brain homogenates.

Preparation of Brain Homogenates

Scrapie-free brain material was obtained from a flock of New Zealandsheep in quarantine.

Scrapie-infected brain material was obtained from a Department ofAgriculture and had been histopathalogically diagnosed as being scrapieinfected.

BSE-infected brain material was obtained via a government AgricultureDepartment and had been histopathalogically certified as being BSEinfected.

BSE-free material was obtained through a private source.

Ha27-30 is brain material obtained from an inbred hamster scrapie model,which has been shown to contain a high level of the scrapie-infectiveagent. It was used as a positive control.

Small samples of infected and uninfected brain were weighed and 10%(w/v) homogenates made up in 10% (v/v) solution of Sarkosyl in 25 mMTris-HCl pH 7.4 (homogenisation buffer). The homogenate was incubated at4° C. for 30 mins and then spun at 6000×g for 30 mins. The supernatantwas collected and the protein content determined using the BCA proteinassay kit (Pierce). The protein concentration was adjusted to 3 mg/mlusing homogenisation buffer.

ELISA (Enzyne-linked immunosorbent assay)

A 8 μM solution of free peptide in PBS was used as the coating antigen.Microtitre plates were coated by adding 50 μl of the antigenconcentration to each well and then incubating for 1 hour at 37° C. toallow binding to occur. Each well was washed 5 times, for 2 minutes,with 300 μl of PBS containing 0.05% Tween 20. After washing, the plateswere blocked by incubating for 1 hour at 37° C. with PBS containing 0.3%Tween 20 and 3% non-fat milk. An aliquot of 50 μl of primary antibody(i.e. antisera) diluted in PBS was added to the appropriate wells andthe plates incubated for 1 hour at 37° C. Plates were washed as before,and then incubated with Horseradish peroxidase conjugated swineanti-rabbit immunoglobulin (anti Ig/HRP) at a dilution of 1:1000 in PBSfor 1 hour at 37° C. The plates were washed and 50 μl of OPD(O-phenylenediamine dihydrochloride substrate (10 mg/ml) in citratebuffer) added to each well and the reaction allowed to proceed at roomtemperature for 10 minutes, before being stopped by the addition ofsulphuric acid. The absorbence of each well was measured at 492 nm usingan ELISA plate reader. The titres were recorded as the dilutions whichgave a positive optical density (OD) reading at least 3 times that ofthe background. The background was taken as the OD readings from wellswhich had not been coated with antigen.

Dot Blot Detection of PrP in Brain Homogenates

The brain homogenates prepared as described earlier were diluted 10-foldin PBS, and 100 μl of homogenates (containing 30 μg total protein) wereapplied to nitrocellulose filters using BRL 96 well vacuum manifold. Thefilters were dried for 1 hour at room temperature. The filters were theneither wet with TBST (10 mM Tris-HCl pH7.4, 150 mM NaCl, 0.05% Tween 20)and PrP detected as described in the immunoblots, or the protein samplefurther treated. This further treatment of the sample included digestionof the protein on the filter using 100 μg/ml proteinase K in TBST for 90minutes at room temperature.

The proteinase K was inactivated by the addition of PMSF(phenylmethylsulphonyl fluoride) to a concentration of 5 mM in TBST.After protein digestion, some samples were also denatured by incubatingthe filters in 6M guanidine HCl containing 5 mM PMSF for 10 minutes. Theguanidine was removed by 3 washes with TBST prior to incubation with theprimary antibody.

Immunoblots. (Western Blots)

SDS-PAGE was performed on the brain homogenates, prepared as describedpreviously, using standard techniques. The samples within the gel weretransferred onto nitrocellulose in a Biorad transblot using TowbinBuffer (25 mM Tris, 190 mM glycine and 0.1% SDS) at 70 mA overnight. Thenitrocellulose filter was blocked with 5% non-fat milk for 30 minutes atroom temperature. The primary antibody (i.e. antisera) diluted in TBSTwas applied for 3 hours at room temperature, the filter washed 3 timesfor 10 minutes in TBST and the filter incubated for 2 hours at roomtemperature with the alkaline phosphatase-conjugated swine anti-rabbitimmunoglobulin diluted at a dilution of 1:2000. After washing, theprotein bands were detected using the NBT/BCIP (nitro-blue tetrazolium;5-bromo-4-chloro-3-indolyl phosphate) substrate (Boehringer Mannheim).

RESULTS

1) Antibody titres: Good antibody titres to the peptides were obtainedin all cases, though the level varied enormously. The peptide which gavethe highest titre, also gave the best results in the dot blots.

2) Dot Blot Data: Uninfected tissue would be expected to contain onlynormal prion protein (PrP^(c)). Infected tissue would be expected tocontain both the normal and the diseased (PrP^(sc)) forms of PrP.

PrP^(c) has a molecular weight of approximately 33-35 kD.

PrP^(sc) has a molecular weight of approximately 27-30 KD and is missingan N-terminal segment that is present in the PrP^(c) form. Otherwise,the amino acid sequence of PrP^(sc) is exactly the same as that ofPrP^(c). Probably the most significant characteristic of PrP^(sc) isresistance to enzyme degradation with proteinase K, a non-specificprotein-digesting enzyme.

When a protein sample is treated with proteinase K any PrP^(c) should becompletely digested. Therefore, in a sample containing only PrP^(c), noPrP of any form will remain after proteinase K treatment. However, in asample containing PrP^(c) and PrP^(sc) (i.e. a diseased sample),PrP^(sc) will remain after treatment.

There are antibodies currently available which recognise PrP^(sc), butthey only recognise the denatured protein. Therefore after proteinase Ktreatment, samples in the dot blot test were treated with guanidine HCl,a denaturing agent, so that such antibodies could be used to detectPrP^(sc).

The data are given in Tables I-V.

Peptide II:

Good titres. Dot blots appear to indicate that some discrimination isoccurring. Negative results were obtained from the Western blots.

Peptide III:

Reasonable titres. Possibly there is recognition of a non-specific(perhaps non-protein) component in the proteinase K and guanidinetreated samples. Negative results were obtained from the Western blots.

Peptide Vb:

Good titres. Although it appears that there might be some discriminationoccurring, the Vb peptide in fact occurs within the N-terminal regionthat is missing in PrP^(sc). One would therefore not expect to see anyrecognition in the infected material treated with proteinase K andquanidine. However, one possible explanation is that the PrP^(c) presentin the infected material has not been completely digested by theproteinase K. Negative results were obtained from the Western blots.

Peptide Vc:

Excellent titres. These results are exactly as expected. As mentionedpreviously, antibodies which recognise PrP^(sc) generally only recognisethe protein in its denatured state. Infected and uninfected samples, aswell as containing PrP^(sc) and/or PrP^(c) in their “native” states,will also contain both PrP forms in various stages of denaturation dueto natural protein turnover within cells. For this reason, antibodieswould be expected to detect all three untreated samples. However,proteinase K treatment will digest PrP^(c) and any partially denaturedPrP^(sc) leading to a loss of antibody recognition in all samplers(assuming the. antibody only recognises denatured PrP). The addition ofguanidine should restore antibody recognition in material which hadoriginally contained PrP^(sc). Western blots showed up the expectedprotein bands at the correct molecular weights.

Peptide VIIIb:

Reasonable titre. There may be recognition of a non-specific component.Negative results were obtained form the Western blots.

Peptides BII & BIII

The titres are reasonable and there are strong positive results fromuntreated normal and infected bovine brain material.

In summary, good anti-peptide titres obtained in all cases, the Westernblots only worked well in the case of peptide Vc, which also gave thehighest titre and the dot blots show that there is some discriminationoccurring between PrP^(c) and PrP^(sc) with peptide Vc. Data frompeptide II also suggests that discrimination is occurring.

TABLE I Results from ovine peptide sequences Pept/ Anti- car- body OvineDOT BLOT rier num- Brain Prot Prot K + West ratio ber Titre MaterialUntrt K Gua Blot II 8:1 93 20,000 infected ++ + + normal ++ − − Ha27-30+/− +/− +/− II 8:1 94 20,000 infected ++ + + normal ++ − − Ha27-30 + + +III 6:1 101 5,000 infected ++ + + normal ++ − + Ha27-30 ++ −− ++ III 6:1102 5,000 infected +++ + + normal +++ +/− +/− Ha27-30 ++ ++ ++ Vc 5:1 97160,000 infected +++ +/− +++ + normal +++ +/− +/− + Ha27-30 +++ ++ +++ +Vc 5:1 98 320,000 infected +++ +/− +++ + normal +++ +/− +/− + Ha27-30+++ +/− +++ +

TABLE II Results from ovine peptide sequences Pept/ Anti- car- bodyOvine DOT BLOT rier num- Brain Prot Prot K + West ratio ber TitreMaterial Untrt K Gua Blot II 8:1 93 20,000 infected ++ + + normal ++ + +Ha27-30 +/− +/− +/− II 8:1 94 20,000 infected ++ + + normal ++ + +Ha27-30 + + + III 6:1 101 5,000 infected ++ + + normal ++ + + Ha27-30 ++++ ++ III 6:1 102 5,000 infected ++ + + normal ++ + + Ha27-30 ++ ++ ++Vc 5:1 97 160,000 infected +++ + ++ normal ++ + + Ha27-30 +++ ++ +++ Vc5:1 98 320,000 infected +++ + ++ normal ++ +/− +/− Ha27-30 +++ +/− +++

TABLE III Results from ovine/bovine peptide sequences Pept/ Anti- DOTBLOT car- body Ovine Prot rier num- Brain Prot K + West ratio ber TitreMaterial Untrt K Gua Blot Vb 6:1 95 50,000 infected ++ + + normal ++ − −Ha27-30 ++ ++ ++ Vb 6:1 96 10,000 infected ++ + + normal ++ − − Ha27-30++ ++ ++ VIIIb 12:1 103 3,000 infected ++ + + normal ++ + + Ha27-30 +++/− +/− VIIIb 12:1 104 3,000 infected + + + normal + + + Ha27-30 + + +

TABLE IV Results from ovine/bovine peptide sequences Pept/ Anti- DOTBLOT car- body Ovine Prot rier num- Brain Prot K + West ratio ber TitreMaterial Untrt K Gua Blot Vb 6:1 95 50,000 infected ++ + + normal ++ + +Ha27-30 ++ ++ ++ Vb 6:1 96 10,000 infected ++ + + normal ++ + + Ha27-30++ ++ ++ VIIIb 12:1 103 3,000 infected ++ + + normal ++ + + Ha27-30 +++/− +/− VIIIb 12:1 104 3,000 infected + + + normal ++ +/− +/−Ha27-30 + + +

TABLE V Results from bovine peptide sequences Pept/ Anti- DOT BLOT car-body Ovine Prot rier num- Brain Prot K + West ratio ber Titre MaterialUntrt K Gua Blot BII 9:1 105 100,000 infected +++ + + normal +++ + +Ha27-30 + + + BII 9:1 106 100,000 infected +++ + + normal +++ + +Ha27-30 + + + BIII 5:1 107 20,000 infected +++ +/− +/− normal +++ +/−+/− Ha27-30 + + + BIII 5:1 108 10,000 infected +++ +/− +/− normal ++++/− +/− Ha27-30 + + +

67 33 amino acids amino acid single linear peptide Modified-site /label=X /note= “X may be absent or present independently of Y and denotes oneor more amino acid(s)” Modified-site 33 /label= Y /note= “Y may beabsent or present independently of X and denotes one or more aminoacid(s) 1 Xaa Met Lys His Val Ala Gly Ala Ala Ala Ala Gly Ala Val ValGly 1 5 10 15 Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg ProLeu Ile 20 25 30 Xaa 33 amino acids amino acid single linear peptideModified-site /label= X /note= ”X may be absent or present independentlyof Y and denotes one or more amino acid(s)“ Modified-site 33 /label= Y/note= ”Y may be absent or present independently of X and denotes one ormore amino acid(s)“ 2 Xaa Met Lys His Met Ala Gly Ala Ala Ala Ala GlyAla Val Val Gly 1 5 10 15 Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala MetSer Arg Pro Ile Ile 20 25 30 Xaa 19 amino acids amino acid linearpeptide Modified-site /label= X /note= ”X may be absent or presentindependently of Y and denotes one or more amino acid(s)“ Modified-site19 /label= Y /note= ”Y may be absent or present independently of X anddenotes one or more amino acid(s)“ 3 Xaa His Val Ala Gly Ala Ala Ala AlaGly Ala Val Val Gly Gly Leu 1 5 10 15 Gly Gly Xaa 19 amino acids aminoacid linear peptide Modified-site /label= X /note= ”Y may be absent orpresent independently of X and denotes one or more amino acid(s)“Modified-site 19 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 4 Xaa Gly GlyLeu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro 1 5 10 15 Leu IleXaa 19 amino acids amino acid single linear peptide Modified-site/label= X /note= ”X may be absent or present independently X of Y anddenotes one or more amino acid(s)“ Modified-site 19 /label= Y /note= ”Ymay be absent or present independently of X and denotes one or moreamino acid(s)“ 5 Xaa His Met Ala Gly Ala Ala Ala Ala Gly Ala Val Val GlyGly Leu 1 5 10 15 Gly Gly Xaa 19 amino acids amino acid single linearpeptide Modified-site /label= X /note= ”X may be absent or presentindependently of Y and denotes one or more amino acid(s)“ Modified-site19 /label= Y /note= ”Y may be absent or present independently of X anddenotes one or more amino acid(s)“ 6 Xaa Gly Gly Leu Gly Gly Tyr Met LeuGly Ser Ala Met Ser Arg Pro 1 5 10 15 Ile Ile Xaa 31 amino acids aminoacid single linear peptide Modified-site /label= X /note= ”X may beabsent or present independently of Y and denotes one or more aminoacid(s)“ Modified-site 31 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 7 Xaa Ser AlaMet Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu 1 5 10 15 Asp ArgTyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln Xaa 20 25 30 31 aminoacids amino acid single linear peptide Modified-site /label= X /note= ”Xmay be absent or present independently of Y and denotes one or moreamino acid(s)“ Modified-site 31 /label= Y /note= ”Y may be absent orpresent independently of X and denotes one or more amino acid(s)“ 8 XaaSer Ala Met Ser Arg Pro Leu Ile His Phe Gly Asn Asp Tyr Glu 1 5 10 15Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn Gln Xaa 20 25 30 31amino acids amino acid single linear peptide Modified-site /label= X/note= ”X may be absent or present independently of Y and denotes one ormore amino acid(s)“ Modified-site 31 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s)“ 9Xaa Ser Ala Met Ser Arg Pro Ile Ile His Phe Gly Ser Asp Tyr Glu 1 5 1015 Asp Arg Tyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln Xaa 20 25 3025 amino acids amino acid single linear peptide Modified-site /label= X/note= ”X may be absent or present independently of Y and denotes one ormore amino acid(s)“ Modified-site 25 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s)“ 10Xaa Ser Ala Met Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu 1 5 1015 Asp Arg Tyr Tyr Arg Glu Asn Met Xaa 20 25 25 amino acids amino acidsingle linear peptide Modified-site /label= X /note= ”X may be absent orpresent independently of Y and denotes one or more amino acid(s)“Modified-site 25 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 11 Xaa Ser AlaMet Ser Arg Pro Leu Ile His Phe Gly Asn Asp Tyr Glu 1 5 10 15 Asp ArgTyr Tyr Arg Glu Asn Met Xaa 20 25 25 amino acids amino acid singlelinear peptide Modified-site /label= X /note= ”X may be absent orpresent independently of Y and denotes one or more amino acid(s)“Modified-site 25 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 12 Xaa Ser AlaMet Ser Arg Pro Ile Ile His Phe Gly Ser Asp Tyr Glu 1 5 10 15 Asp ArgTyr Tyr Arg Glu Asn Met Xaa 20 25 31 amino acids amino acid singlelinear peptide Modified-site /label= X /note= ”X may be absent orpresent independently of Y and denotes one or more amino acid(s)“Modified-site 31 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 13 Xaa Asn MetHis Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp 1 5 10 15 Gln TyrSer Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 30 31 aminoacids amino acid single linear peptide Modified-site /label= X /note= ”Xmay be absent or present independently of Y and denotes one or moreamino acid(s)“ Modified-site 31 /label= Y /note= ”Y may be absent orpresent independently of X and denotes one or more amino acid(s)“ 14 XaaAsn Met Tyr Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp 1 5 10 15Arg Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 30 31amino acids amino acid single linear peptide Modified-site /label= X/note= ”X may be absent or present independently of Y and denotes one ormore amino acid(s)“ Modified-site 31 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s)“ 15Xaa Asn Met His Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Met Asp 1 5 1015 Glu Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 3028 amino acids amino acid single linear peptide Modified-site /label= X/note= ”X may be absent or present independently of Y and denotes one ormore amino acid(s)“ Modified-site 28 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s)“ 16Xaa Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp Gln Tyr Ser 1 5 1015 Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 28 amino acidsamino acid single linear peptide Modified-site /label= X /note= ”X maybe absent or present independently of Y and denotes one or more aminoacid(s)“ Modified-site 28 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s) “ 17 Xaa ArgTyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp Arg Tyr Ser 1 5 10 15 AsnGln Asn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 28 amino acids aminoacid single linear peptide Modified-site /label= X /note= ”X may beabsent or present independently of Y and denotes one or more aminoacid(s)“ Modified-site 28 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 18 Xaa Arg TyrPro Asn Gln Val Tyr Tyr Arg Pro Met Asp Glu Tyr Ser 1 5 10 15 Asn GlnAsn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 31 amino acids amino acidsingle linear peptide Modified-site /label= X /note= ”X may be absent orpresent independently of Y and denotes one or more amino acid(s)“Modified-site 31 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 19 Xaa Tyr TyrGln Arg Gly Ala Ser Val Ile Leu Phe Ser Ser Pro Pro 1 5 10 15 Val IleLeu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly Xaa 20 25 30 31 aminoacids amino acid single linear peptide Modified-site /label= X /note= ”Xmay be absent or present independently of Y and denotes one or moreamino acid(s)“ Modified-site 31 /label= Y /note= ”Y may be absent orpresent independently of X and denotes one or more amino acid(s)“ 20 XaaTyr Tyr Gln Arg Gly Ser Ser Met Val Leu Phe Ser Ser Pro Pro 1 5 10 15Val Ile Leu Leu Ile Ser Phe Leu Ile Phe Leu Ile Val Gly Xaa 20 25 30 19amino acids amino acid single linear peptide Modified-site /label= X/note= ”X may be absent or present independently of Y and denotes one ormore amino acid(s)“ Modified-site 19 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s)“ 21Xaa Gly Ala Ser Val Ile Leu Phe Ser Ser Pro Pro Val Ile Leu Leu 1 5 1015 Ile Ser Xaa 19 amino acids amino acid single linear peptideModified-site /label= X /note= ”X may be absent or present independentlyof Y and denotes one or more amino acid(s)“ Modified-site 19 /label= Y/note= ”Y may be absent or present independently of X and denotes one ormore amino acid(s)“ 22 Xaa Gly Ser Ser Met Val Leu Phe Ser Ser Pro ProVal Ile Leu Leu 1 5 10 15 Ile Ser Xaa 33 amino acids amino acid singlelinear peptide Modified-site /label= X /note= ”X may be absent orpresent independently of Y and denotes one or more amino acid(s)“Modified-site 33 /label= Y /note= ”Y may be absent or presentindependently of X and denotes one or more amino acid(s)“ 23 Xaa Pro GlyGly Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln 1 5 10 15 Gly SerPro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp 20 25 30 Xaa 18amino acids amino acid single linear peptide Modified-site /label= X/note= ”X may be absent or present independently of Y and denotes one ormore amino acid(s)“ Modified-site 18 /label= Y /note= ”Y may be absentor present independently of X and denotes one or more amino acid(s)“ 24Xaa Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro 1 5 1015 His Xaa 30 amino acids amino acid single linear peptide Modified-site/label= X /note= ”X may be absent or present independently of Y anddenotes one or more amino acid(s) Modified-site 30 /label= Y /note= “Ymay be absent or present independently of X and denotes one or moreamino acid(s)” 25 Xaa Gly Gly Gly Trp Gly Gln Gly Gly Thr His Gly GlnTrp Asn Lys 1 5 10 15 Pro Ser Lys Pro Lys Thr Asn Met Lys His Val AlaGly Xaa 20 25 30 33 amino acids amino acid single linear peptideModified-site /label= X /note= “X may be absent or present independentlyof Y and denotes one or more amino acid(s)” Modified-site 33 /label= Y/note= “Y may be absent or present independently of X and denotes one ormore amino acid(s)” 26 Xaa Pro Gly Gly Gly Trp Asn Thr Gly Gly Ser ArgTyr Pro Gly Gln 1 5 10 15 Gly Ser Pro Gly Gly Asn Arg Tyr Pro Pro GlnGly Gly Gly Gly Trp 20 25 30 Xaa 18 amino acids amino acid single linearpeptide Modified-site /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site18 /label= Y /note= “Y may be absent or present independently of X anddenotes one or more amino acid(s)” 27 Xaa Gly Gly Gly Trp Gly Gln ProHis Gly Gly Gly Trp Gly Gln Pro 1 5 10 15 His Xaa 30 amino acids aminoacid single linear peptide Modified-site /label= X /note= “X may beabsent or present independently of Y and denotes one or more aminoacid(s)” Modified-site 30 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 28 Xaa Gly GlyGly Trp Gly Gln Gly Gly Ser His Ser Gln Trp Asn Lys 1 5 10 15 Pro SerLys Pro Lys Thr Asn Met Lys His Val Ala Gly Xaa 20 25 30 33 amino acidsamino acid single linear peptide Modified-site /label= X /note= “X maybe absent or present independently of Y and denotes one or more aminoacid(s)” Modified-site 33 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s) ” 29 Xaa ProGly Gly Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln 1 5 10 15 GlySer Pro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp 20 25 30 Xaa18 amino acids amino acid single linear peptide Modified-site /label= X/note= “X is absent or present independently of Y and denotes one ormore amino acid(s)” Modified-site 18 /label= Y /note= “Y may be absentor present independently of X and denotes one or more amino acid(s)” 30Xaa Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro 1 5 1015 His Xaa 31 amino acids amino acid single linear peptide Modified-site/label= X /note= “X may be absent or present independently of Y anddenotes one or more amino acid(s)” Modified-site 31 /label= Y /note= “Ymay be absent or present independently of X and denotes one or moreamino acid(s)” 31 Xaa Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His SerGln Trp Asn 1 5 10 15 Lys Pro Ser Lys Pro Lys Thr Asn Met Lys His MetAla Gly Xaa 20 25 30 33 amino acids amino acid single linear peptideModified-site /label= X /note= “X may be absent or present independentlyof Y and denotes one or more amino acid(s)” Modified-site 33 /label= Y/note= “Y may be absent or present independently of X and denotes one ormore amino acid(s)” 32 Xaa Asn Phe Val His Asp Cys Val Asn Ile Thr ValLys Glu His Thr 1 5 10 15 Val Thr Thr Thr Thr Lys Gly Glu Asn Phe ThrGlu Thr Asp Ile Lys 20 25 30 Xaa 22 amino acids amino acid single linearpeptide Modified-site /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site22 /label= Y /note= “Y may be absent or present independently of X anddenotes one or more amino acid(s)” 33 Xaa Met Cys Ile Thr Gln Tyr GlnArg Glu Ser Gln Ala Tyr Tyr Gln 1 5 10 15 Arg Gly Ala Ser Val Xaa 20 33amino acids amino acid single linear peptide Modified-site /label= X/note= “X may be absent or present independently of Y and denotes one ormore amino acid(s)” Modified-site 33 /label= Y /note= “Y is absent orpresent independently of X and denotes one or more amino acid(s)” 34 XaaAsn Phe Val His Asp Cys Val Asn Ile Thr Val Lys Gln His Thr 1 5 10 15Val Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Ile Lys 20 25 30Xaa 22 amino acids amino acid single linear peptide Modified-site/label= X /note= “X may be absent or present independently of Y anddenotes one or more amino acid(s)” Modified-site 22 /label= Y /note= “Ymay be absent or present independently of X and denotes one or moreamino acid(s)” 35 Xaa Met Cys Ile Thr Gln Tyr Gln Arg Glu Ser Gln AlaTyr Tyr Gln 1 5 10 15 Arg Gly Ala Ser Val Xaa 20 33 amino acids aminoacid single linear peptide Modified-site /label= X /note= “X may beabsent or present independently of Y and denotes one or amino acid(s)”Modified-site 33 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 36 Xaa Asn PheVal His Asp Cys Val Asn Ile Thr Ile Lys Gln His Thr 1 5 10 15 Val ThrThr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp Val Lys 20 25 30 Xaa 22amino acids amino acid single linear peptide Modified-site /label= X/note= “X may be absent or present independently of Y and denotes one ormore amino acid(s)” Modified-site 22 /label= Y /note= “Y may be absentor present independently of X and denotes one or more amino acid(s)” 37Xaa Met Cys Ile Thr Gln Tyr Glu Arg Glu Ser Gln Ala Tyr Tyr Gln 1 5 1015 Arg Gly Ser Ser Met Xaa 20 5 amino acids amino acid single linearpeptide 38 Gly Gly Gly Gly Gly 1 5 6 amino acids amino acid singlelinear peptide 39 Gly Pro Gly Pro Gly Pro 1 5 7 amino acids amino acidsingle linear peptide 40 Gly Ser Ala Gly Ser Gly Ala 1 5 26 amino acidsamino acid single linear peptide 41 Ala Met Ser Arg Pro Leu Ile His PheGly Ser Asp Tyr Glu Asp Arg 1 5 10 15 Tyr Tyr Arg Glu Asn Met His ArgGly Cys 20 25 21 amino acids amino acid single linear peptide 42 Ser AlaMet Ser Arg Pro Leu Ile His Phe Gly Asn Asp Tyr Glu Asp 1 5 10 15 ArgTyr Tyr Gly Cys 20 21 amino acids amino acid single linear peptide 43Ser Ala Met Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu Asp 1 5 1015 Arg Tyr Tyr Gly Cys 20 27 amino acids amino acid single linearpeptide 44 Asn Met Tyr Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val AspArg 1 5 10 15 Tyr Ser Asn Gln Asn Asn Phe Val His Gly Cys 20 25 27 aminoacids amino acid single linear peptide 45 Asn Met His Arg Tyr Pro AsnGln Val Tyr Tyr Arg Pro Val Asp Gln 1 5 10 15 Tyr Ser Asn Gln Asn AsnPhe Val His Gly Cys 20 25 26 amino acids amino acid single linearpeptide 46 Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly GlyTrp 1 5 10 15 Gly Gln Pro His Gly Gly Gly Trp Gly Cys 20 25 24 aminoacids amino acid single linear peptide 47 Gly Gln Gly Gly Ser His SerGln Trp Asn Lys Pro Ser Lys Pro Lys 1 5 10 15 Thr Asn Met Lys His ValGly Cys 20 15 amino acids amino acid single linear peptide 48 Cys IleThr Gln Tyr Gln Arg Glu Ser Gln Ala Tyr Tyr Gln Arg 1 5 10 15 28 aminoacids amino acid single linear peptide 49 Gly Gly Trp Asn Thr Gly GlySer Arg Tyr Pro Gly Gln Gly Ser Pro 1 5 10 15 Gly Gly Asn Arg Tyr ProPro Gln Gly Gly Gly Cys 20 25 23 amino acids amino acid single linearpeptide 50 Val Asn Ile Thr Val Lys Gln His Thr Val Thr Thr Thr Thr LysGly 1 5 10 15 Glu Asn Phe Thr Glu Gly Cys 20 29 amino acids amino acidsingle linear peptide 51 Lys His Met Ala Gly Ala Ala Ala Ala Gly Ala ValVal Gly Gly Leu 1 5 10 15 Gly Gly Tyr Met Leu Gly Ser Ala Met Ser ArgGly Cys 20 25 33 amino acids amino acid single linear peptideModified-site /label= X /note= “X may be absent or present independentlyof Y and denotes one or amino acid(s)” Modified-site /label= X /note= “X= either Met, Leu or Phe” Modified-site /label= X /note= “X = Met orVal” Modified-site 11 /label= X /note= “X = Ala or absent” Modified-site31 /label= X /note= “X = either Leu, Ile or Met” Modified-site 32/label= X /note= “X = Leu, Ile or Met” Modified-site 33 /label= Y /note=“Y may be absent or present independently of X and denotes one or moreamino acid(s)” 52 Xaa Xaa Lys His Xaa Ala Gly Ala Ala Ala Xaa Gly AlaVal Val Gly 1 5 10 15 Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met SerArg Pro Xaa Xaa 20 25 30 Xaa 31 amino acids amino acid single linearpeptide Modified-site /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site/label= X /note= “X = either Leu, Ile, or Met” Modified-site /label= X/note= “X = either Leu, Ile or Met” Modified-site 13 /label= X /note= “X= Asn or Ser” Modified-site 15 /label= X /note= “X = Tyr or Trp”Modified-site 25 /label= X /note= “X = either His, Tyr or Asn”Modified-site 31 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 53 Xaa Ser AlaMet Ser Arg Pro Xaa Xaa His Phe Gly Xaa Asp Xaa Glu 1 5 10 15 Asp ArgTyr Tyr Arg Glu Asn Met Xaa Arg Tyr Pro Asn Gln Xaa 20 25 30 31 aminoacids amino acid single linear peptide Modified-site /label= X /note= “Xmay be absent or present independently of Y and denotes one or moreamino acid(s)” Modified-site /label= X /note= “X = either His, Tyr orAsn” Modified-site 15 /label= X /note= “X = Val or Met” Modified-site 17/label= X /note= “X = either Gln, Glu or Arg” Modified-site 19 /label= X/note= “X = Ser or Asn” Modified-site 31 /label= Y /note= “Y may beabsent or present independently of X and denotes one or more aminoacid(s)” 54 Xaa Asn Met Xaa Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro XaaAsp 1 5 10 15 Xaa Tyr Xaa Asn Gln Asn Asn Phe Val His Asp Cys Val AsnXaa 20 25 30 33 amino acids amino acid single linear peptideModified-site /label= X /note= “X may be absent or present independentlyof Y and denotes one or more amino acid(s)” Modified-site /label= X/note= “X = Asp or Gln” Modified-site /label= X /note= “X = Gly orabsent” Modified-site /label= X /note= “X = Gly or Arg” Modified-site/label= X /note= “X = Ala or Ser” Modified-site 10 /label= X /note= “X =Ser or absent” Modified-site 11 /label= X /note= “X = Ala, Thr, Met orVal” Modified-site 12 /label= X /note= “X = Val or Ile” Modified-site 30/label= X /note= “X= Ile or Met” Modified-site 33 /label= Y /note= “Ymay be absent or present independently of X and denotes one or moreamino acid(s)” 55 Xaa Tyr Tyr Xaa Xaa Arg Xaa Xaa Ser Xaa Xaa Xaa LeuPhe Ser Ser 1 5 10 15 Pro Pro Val Ile Leu Leu Ile Ser Phe Leu Ile PheLeu Xaa Val Gly 20 25 30 Xaa 33 amino acids amino acid single linearpeptide Modified-site /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site/label= X /note= “X = Gly or absent” Modified-site 30 /label= X /note=“X = Gly or absent” Modified-site 31 /label= X /note= “X = Gly or Thr”Modified-site 33 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 56 Xaa Pro GlyGly Xaa Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln 1 5 10 15 Gly SerPro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly Xaa Xaa Trp 20 25 30 Xaa 20amino acids amino acid single linear peptide Modified-site /label= X/note= “X may be absent or present independently of Y and denotes one ormore amino acid(s)” Modified-site /label= X /note= “X = Gly or absent”Modified-site /label= X /note= “X = Gly or Thr” Modified-site 14 /label=X /note= “X = Gly or absent” Modified-site 20 /label= Y /note= “Y may beabsent or present independently of X and denotes on or more aminoacid(s)” 57 Xaa Gly Gly Xaa Xaa Trp Gly Gln Pro His Gly Gly Gly Xaa TrpGly 1 5 10 15 Gln Pro His Xaa 20 31 amino acids amino acid single linearpeptide Modified-site /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site10 /label= X /note= “X = Gly or absent” Modified-site 11 /label= X/note= “X = Thr or Ser” Modified-site 13 /label= X /note= “X = eitherGly, Ser or Asn” Modified-site 19 /label= X /note= “X = Asn or Ser”Modified-site 24 /label= X /note= “X = Asn or Ser” Modified-site 25/label= X /note= “X = either Met, Leu or Phe” Modified-site 28 /label= X/note= “X = Val or Met” Modified-site 31 /label= Y /note= “Y may beabsent or present independently of X and denotes one or more aminoacid(s)” 58 Xaa Gly Gly Gly Trp Gly Gln Gly Gly Xaa Xaa His Xaa Gln TrpAsn 1 5 10 15 Lys Pro Xaa Lys Pro Lys Thr Xaa Xaa Lys His Xaa Ala GlyXaa 20 25 30 18 amino acids amino acid single linear peptideModified-site /label= X /note= “X may be absent or present independentlyof Y and denotes one or more amino acid(s)” Modified-site /label= X/note= “X = Ala or Thr” Modified-site /label= X /note= “X = either Ser,Pro or Thr” Modified-site /label= X /note= “X = Trp or Arg”Modified-site /label= X /note= “X = either, Ala, Ser, Pro, and Thr”Modified-site 10 /label= X /note= “X = Ala or Thr” Modified-site 11/label= X /note= “X = either Ser, Pro or Thr” Modified-site 13 /label= X/note= “X = Trp or Arg” Modified-site 17 /label= X /note= “X = eitherAla, Ser, Pro, or Thr” Modified-site 18 /label= Y /note= “Y may beabsent or present independently of X and denotes one or more aminoacid(s)” 59 Xaa Xaa Xaa Trp Xaa Trp Leu Gly Xaa Xaa Xaa Trp Xaa Trp LeuGly 1 5 10 15 Xaa Xaa 18 amino acids amino acid single linear peptideModified-site /label= X /note= “X may be absent or present independentlyof Y and denotes one or more amino acid(s)” Modified-site /label= X/note= “X = Ser or Asn” Modified-site /label= X /note= “X = either Pro,Leu or His” Modified-site /label= X /note= “X = Val or Glu”Modified-site /label= X /note= “X = either Val, Ala, Asp or Gly”Modified-site 10 /label= X /note= “X = Ser or Asn” Modified-site 11/label= X /note= “X = either Pro, Leu or His” Modified-site 13 /label= X/note= “X = Val or Glu” Modified-site 17 /label= X /note= “X = eitherVal, Ala, Asp or Gly” Modified-site 18 /label= Y /note= “Y may be absentor present independently of X and denotes one or more amino acid(s)” 60Xaa Xaa Xaa Met Xaa Val Ala Gly Xaa Xaa Xaa Met Xaa Val Ala Gly 1 5 1015 Xaa Xaa 33 amino acids amino acid single linear peptide Modified-site/label= X /note= “X may be absent or present independently of Y anddenotes one or more amino acid(s)” Modified-site 12 /label= X /note= “X= Ile or Val” Modified-site 14 /label= X /note= “X = Gln or Glu”Modified-site 18 /label= X /note= “X = Val or Thr” Modified-site 31/label= X /note= “X = Val or Ile” Modified-site 33 /label= Y /note= “Ymay be absent or present independently of X and denotes one or moreamino acid(s)” 61 Xaa Asn Phe Val His Asp Cys Val Asn Ile Thr Xaa LysXaa His Thr 1 5 10 15 Val Xaa Thr Thr Thr Lys Gly Glu Asn Phe Thr GluThr Asp Xaa Lys 20 25 30 Xaa 24 amino acids amino acid single linearpeptide Modified-site /label= X /note= “X may be absent or presentindependently of Y and denotes one or more amino acid(s)” Modified-site/label= X /note= “X = either Ile, Thr or Val” Modified-site /label= X/note= “X = Gln or Glu” Modified-site /label= X /note= “X = Arg or Lys”Modified-site 16 /label= X /note= “X = Asp or Gln” Modified-site 17/label= X /note= “X = Gly or absent” Modified-site 19 /label= X /note=“X = Gly or Arg” Modified-site 20 /label= X /note= “X = Ala or Ser”Modified-site 22 /label= X /note= “X = Ser or absent” Modified-site 23/label= X /note= “X = either Ala, Thr, Met or Val” Modified-site 24/label= Y /note= “Y may be absent or present independently of X anddenotes one or more amino acid(s)” 62 Xaa Met Cys Xaa Thr Gln Tyr XaaXaa Glu Ser Gln Ala Tyr Tyr Xaa 1 5 10 15 Xaa Arg Xaa Xaa Ser Xaa XaaXaa 20 18 amino acids amino acid single linear peptide Modified-site/label= X /note= “X may be absent or present independently of Y anddenotes one or more amino acid(s)” Modified-site /label= X /note= “X =Met or Val” Modified-site /label= X /note= “X = Ala or absent”Modified-site 18 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 63 Xaa His XaaAla Gly Ala Ala Ala Xaa Gly Ala Val Val Gly Gly Leu 1 5 10 15 Gly Xaa 19amino acids amino acid single linear peptide Modified-site /label= X/note= “X may be absent or present independently of Y and denotes one ormore amino acid(s)” Modified-site 17 /label= X /note= “X = either Leu,Ile, or Met” Modified-site 18 /label= X /note= “X = either Leu, Ile, orMet” Modified-site 19 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 64 Xaa Gly GlyLeu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro 1 5 10 15 Xaa XaaXaa 25 amino acids amino acid single linear peptide Modified-site/label= X /note= “X may be absent or present independently of Y anddenotes one or more amino acid(s)” Modified-site /label= X /note= “X =either Leu, Ile or Met” Modified-site /label= X /note= “X = either Leu,Ile or Met” Modified-site 13 /label= X /note= “X = Asn or Ser”Modified-site 15 /label= X /note= “X = Tyr or Trp” Modified-site 25/label= /note= “Y may be absent or present independently of X anddenotes one or more amnio acid(s)” 65 Xaa Ser Ala Met Ser Arg Pro XaaXaa His Phe Gly Xaa Asp Xaa Glu 1 5 10 15 Asp Arg Tyr Tyr Arg Glu AsnMet Xaa 20 25 28 amino acids amino acid single linear peptideModified-site /label= X /note= “X may be absent or present independentlyof Y and denotes one or more amino acid(s)” Modified-site 12 /label= X/note= “X = Val or Met” Modified-site 14 /label= X /note= “X = eitherGln, Glu or Arg” Modified-site 16 /label= X /note= “X = Ser or Asn”Modified-site 28 /label= Y /note= “Y may be absent or presentindependently of X and denotes one or more amino acid(s)” 66 Xaa Arg TyrPro Asn Gln Val Tyr Tyr Arg Pro Xaa Asp Xaa Tyr Xaa 1 5 10 15 Asn GlnAsn Asn Phe Val His Asp Cys Val Asn Xaa 20 25 20 amino acids amino acidsingle linear peptide Modified-site /label= X /note= “X may be absent orpresent independently of Y and denotes one or more amino acid(s)”Modified-site /label= X /note= “X = Gly or Arg” Modified-site /label= X/note= “X = Ala or Ser” Modified-site /label= X /note= “X = Ser orabsent” Modified-site /label= X /note= “X = either Ala, Thr, Met or Val”Modified-site /label= X /note= “X = Val or Ile” Modified-site 20 /label=Y /note= “Y may be absent or present independently of X and denotes oneor more amino acid(s)” 67 Xaa Xaa Xaa Ser Xaa Xaa Xaa Leu Phe Ser SerPro Pro Val Ile Leu 1 5 10 15 Leu Ile Ser Xaa 20

What is claimed is:
 1. A method for detecting prion proteins ofantibodies against prion proteins which comprises incubating a samplewith an antibody or antigen binding fragment that will cross react withprion proteins in more than one species and which specifically binds toa synthetic polypeptide comprising a sequence according to generalFormula (I):X-(R₁-Lys-His-R₂)-Ala-Gly-Ala-Ala-Ala-R₃-Gly-Ala-Val-Val-Gly—Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-R₄-R₅)-Y(Formula I (SEQ ID NO: 52))  wherein: R₁ is an amino acid residueselected from Met, Leu and Phe; R₂ is either Met or Val; R₃ is Ala or isabsent; R₄ and R₅ are independently an amino acid residue selected fromLeu, Ile and Met; one or more residues within the parentheses arepresent or absent with the proviso that if they are present they areattached to the rest of the peptide in sequence; and X and Y are eachindependently absent or one or more additional amino acid residues, withthe proviso that when present neither X nor Y provide or form part of anantigenic property of the prion protein which, in the correspondingportion of sequence of a natural prion protein, is contiguous with thesequence to which X and Y are attached; and assaying the incubatedsamples to detect the prion proteins or antibodies.
 2. The method ofclaim 1 wherein the antibody or antigen binding fragment specificallybinds to a sub-fragment having the sequence: Seq. I.D. No: 63X-(His-R₂-Ala-Gly)-Ala-Ala-Ala-R₃-Gly-Ala-Val-Val-(Gly-Gly-Leu-Gly)-Y;or Seq. I.D. No: 64X-(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-R₄-R₅)-Ywherein R₂, R₃, R₄, R₅, X and Y are as defined in Formula I and one ormore residues in the parentheses are absent or present as in Formula I.3. The method of claim 2 wherein the antibody or antigen bindingfragment specifically binds to the synthetic polypeptide: Seq. I.D. No:3 i)X-(His-Val-Ala-Gly)-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-(Gly-Leu-Gly-Gly)-Y;Seq. I.D. No: 4 ii)X-(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Leu-Ile)-YSeq. I.D. No: 5 i)X-(His-Met-Ala-Gly)-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-(Gly-Leu-Gly-Gly)-Y;or Seq. I.D. No: 6 ii)X-(Gly-Gly-Leu-Gly)-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Ile-Ile)-Y.4. The method of claim 3 wherein the antibody or antigen bindingfragment is provided in a kit.
 5. The method of claim 3 wherein theassaying step comprises using an immunoassaying technique that includesa sandwich assay, a competitive assay, a non-competitive assay, ordirect or indirect labelling.
 6. The method of claim 2 wherein theantibody or antigen binding fragment is provided in a kit.
 7. The methodof claim 2 wherein the assaying step comprises using an immunoassayingtechnique that includes a sandwich assay, a competitive assay, anon-competitive assay, or direct or indirect labelling.
 8. The method ofclaim 1 wherein the antibody or antigen binding fragment is provided ina kit.
 9. The method of claim 1 wherein the assaying step comprisesusing an immunoassaying technique that includes a sandwich assay, acompetitive assay, a non-competitive assay, or direct or indirectlabelling.
 10. The method of claim 1 wherein the antibodies aredeveloped using an immunogen that is a combination of at least two ofbovine, ovine, or human prion proteins.
 11. The method of claim 10wherein the antibodies are developed using an immunogen that is acombination of bovine, ovine, and human prion proteins.
 12. The methodof claim 1 wherein the polypeptide is conjugated to a carrier beforebeing incubated with the sample.
 13. A method for detecting prionproteins or antibodies against prion proteins which comprises incubatinga sample with an antibody or antigen binding fragment that will crossreact with prion proteins in more than one species and whichspecifically binds to the synthetic polypeptide: Seq. I.D. No: 1X-(Met-Lys-His-Val)-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Leu-Ile)-Y;or Seq. I.D. No: 2X-(Met-Lys-His-Met)-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-(Arg-Pro-Ile-Ile)-Y;and assaying the incubated samples to detect the prion proteins orantibodies.
 14. The method of claim 13 wherein the antibody or antigenbinding fragment is provided in a kit.
 15. The method of claim 13wherein the assaying step comprises using an immunoassaying techniquethat includes a sandwich assay, a competitive assay, a non-competitiveassay, or direct or indirect labelling.
 16. A method for detecting prionproteins or antibodies against prion proteins which comprises incubatinga sample with an antibody or antigen binding fragment that will crossreact with prion proteins in more than one species and whichspecifically binds to the synthetic polypeptide: Seq. I.D. No: 51Lys-His-Met-Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala-Val-Val-Gly-Gly-Leu-Gly-Gly-Tyr-Met-Leu-Gly-Ser-Ala-Met-Ser-Arg-Gly-Cys;and assaying the incubated samples to detect the prion proteins orantibodies.
 17. The method of claim 16 wherein the antibody or antigenbinding fragment is provided in a kit.
 18. The method of claim 16wherein the assaying step comprises using an immunoassaying techniquethat includes a sandwich assay, a competitive assay, a non-competitiveassay, or direct or indirect labelling.